The present invention relates to the determination of immunoreactive polypeptides capable of reacting specifically with the antibodies of patients suffering from multiple sclerosis (MS), and the use of these polypeptides.
The Applicant has defined a polypeptide capable of reacting specifically with the antibodies of patients suffering from multiple sclerosis, and which must additionally meet at least any one of the following definitions, provided that said polypeptide is different from the polypeptides having one of the following sequences: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27:
its peptide sequence comprises at least one sequence chosen from SEQ ID NO: 1 to SEQ ID NO: 19, and their equivalent sequences;
its peptide sequence consists of a sequence chosen from SEQ ID NO: 1 to SEQ ID NO: 19, and their equivalent sequences; preferably, it is chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 19;
it comprises a sequence equivalent to SEQ ID NO: 11, said equivalent sequence exhibiting, for a succession of 8 contiguous amino acids, at least 35% (which corresponds to at least 3 amino acids), preferably 50% (which corresponds to at least 4 amino acids) identity, and/or at least 60% (which corresponds to about at least 5 amino acids), preferably 75% (which corresponds to at least 6 amino acids) homology with a sequence of the foot-and-mouth disease virus SAT3 protein, said polypeptide being different from the whole or part of said SAT3 protein;
it comprises a sequence equivalent to SEQ ID NO: 13 exhibiting, for a succession of 12 contiguous amino acids, at least 40%, preferably 50% identity, and/or at least 55%, preferably 65% homology with a sequence p30/p10/5xe2x80x2v-fsm of the coding region of the feline sarcoma virus (FSV) [NCBI reference gi/554646], said polypeptide being different from the whole or part of said sequence p30/p10/5xe2x80x2v-fsm.
Moreover, the work by the Applicant, in the search for an etiology of MS, has led to the discovery of the existence of at least one pathological and/or infective agent, the retrovirus MSRV-1, in particular associated with multiple sclerosis.
The techniques for the culture and detection of retroviral material which were used in the work carried out by the Applicant on a multiple sclerosis (MS) associated agent are described in French Patent Applications 92 04322, 92 13447, 92 13443, 92 01529, 94 01530, 94 01531, 94 01532 and in the publication by H. PERRON et al. (Res. Virol. 1989; 140, 551-561) (the content of which is incorporated by reference into the present description).
This retrovirus may be derived from a viral strain chosen from the strains called, respectively, POL-2, deposited on 22.07.1992 at the ECACC under the accession number V92072202, and MS7PG deposited on 08.01.93 at the ECACC under the accession number V93010816, or produced by a cell line chosen from the lines called, respectively, PLI-2 deposited on 22.07.1992 at the ECACC under the accession number 92072201, and LM7PC deposited on 08.01.93 at the ECACC under the accession number 93010817.
Among the polypeptides of the invention, the Applicant has determined a polypeptide (generically called pxMSRV-1 in the remainder of the description) capable of reacting specifically with at least one biological fluid from a patient in whom the MSRV-1 viral sequences have been detected. According to the invention, this polypeptide possesses a peptide sequence which comprises at least one sequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and their equivalent sequences, said polypeptide being different from the polypeptides having any one of the following sequences: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 25. Preferably, the sequence of the polypeptide pxMSRV-1 consists of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
Before disclosing the invention in greater detail, various terms used in the description and the claims are defined below.
xe2x80x9cPolypeptidexe2x80x9d designates a peptide, in the isolated state, exhibiting a succession of a variable number of amino acids, such as an oligopeptide, a protein, a fusion protein, a fusion peptide, a synthetic peptide. A polypeptide may be obtained by various techniques well known to persons skilled in the art, and in particular by chemical synthesis or by genetic recombination techniques. The polypeptides according to the invention may be obtained by methods of conventional synthesis, for example with an automated peptide synthesizer, or by genetic engineering techniques comprising the insertion of a DNA sequence encoding said polypeptide into an expression vector such as a plasmid or a virus, and the transformation of cells with this expression vector and culture of these cells.
A polypeptide of the invention advantageously contains at most 50 amino acids, preferably at most 30 amino acids, or better still at most 20 amino acids, or even at most 15 amino acids.
xe2x80x9cPeptide sequence equivalent to a reference peptide sequence is understood to mean an amino acid sequence modified by insertion and/or deletion and/or substitution and/or extension and/or shortening and/or chemical modification of one or more amino acids, as long as these modifications substantially preserve or even develop the immunoreactive properties of said reference peptide sequence. Advantageously, said equivalent sequence exhibits, for at least a succession of 6 amino acids, a percentage identity of at least 40%, preferably of at least 50%, or better still of at least 60% or even 70%, with said reference sequence. This percentage identity is calculated according to the following steps: a succession of 6 contiguous amino acids of the sequence analyzed is compared with a succession of 6 contiguous amino acids of the reference sequence, the amino acids which are common between the analyzed sequence and the reference sequence, located at the same position, are determined and the percentage identity is deduced.
xe2x80x9cEquivalent sequencexe2x80x9d is thus understood to mean in particular the sequences in which one or more amino acids are substituted by one or more other amino acids; the sequences in which one or more amino acids of the L series are replaced by an amino acid of the D series, and vice versa; the sequences in which a modification of the side chains of the amino acids, such as an acetylation of the amine functions, a carboxylation of the thiol functions, an esterification of the carboxyl functions, is introduced; a modification of the peptide bonds such as, for example, the carba, retro, inverse, retro-inverse, reduced and methyleneoxy bonds.
The equivalence of a peptide sequence relative to a reference peptide sequence may be defined by its identity or its homology, expressed as a percentage, with said reference sequence. This percentage is determined, for a succession of a given number of contiguous amino acids, by aligning the two sequences, moving one relative to the other, and comparing the amino acids in the two sequences. The percentage identity is determined from the number of amino acids which are identical to the amino acids of the reference sequence, at the same position. The percentage homology is determined from the number of amino acids which are equivalent to amino acids of the reference sequence, at the same position. Using the BLAST program (BLAST p matrix Blosum62) publicly available (on the Internet, at the National Center for Biotechnology Information (NCBI), Bethesda, USA), persons skilled in the art are in a position to know if the sequence which they have selected exhibits the percentage homology required according to the invention, compared with the reference sequence. In accordance with the BLAST program, two amino acids are equivalent if they possess similar physicochemical properties, such as hydrophilicity, isoelectric point.
Viral sequence of the MSRV-1 virus is understood to mean in particular all the nucleotide sequences described in French Patent Applications 92 04322, 92 13447, 92 13443, 92 01529, 94 01530, 94 01531, 94 01532, 95 09643, in the name of the Applicant.
Binding of antibodies is understood to mean the separation, isolation, detection and/or quantification of these antibodies, the enrichment of an antibody fraction, according to a specific or aspecific binding method, qualitatively and/or quantitatively.
xe2x80x9cPolynucleotidexe2x80x9d is understood to mean either a DNA sequence or an RNA sequence or a cDNA sequence resulting from the reverse transcription of an RNA sequence, of natural or synthetic origin, with or without modified bases.
The invention additionally relates to:
a reagent for the detection of multiple sclerosis in a patient and/or the monitoring of a patient suffering from multiple sclerosis, comprising at least one, or consisting of a, polypeptide capable of reacting specifically with the antibodies of patients suffering from multiple sclerosis (MS) and whose peptide sequence comprises at least one sequence chosen from SEQ ID NO: 1 to SEQ ID NO: 19, and their equivalent sequences, said polypeptide being optionally labeled; as well as a kit comprising this reagent, the latter being supported on a support which is immunologically compatible with said reagent;
a reagent for the detection of an MSRV-1 virus infection comprising at least one, or consisting of a, polypeptide pxMSRV-1 capable of reacting with at least one biological fluid from a patient in whom MSRV-1 viral sequences have been detected and whose peptide sequence comprises at least one, or consists of a, sequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and their equivalent sequences, said polypeptide being optionally labeled; as well as a kit comprising this abovementioned reagent optionally supported on a support which is immunologically compatible with said reagent;
Advantageously, a reagent of the invention comprises at least two different polypeptides as defined above and in particular three polypeptides as defined above. In a particularly preferred form, a reagent comprises the three polypeptides whose peptide sequence each consists of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 16, respectively.
a method of binding, in a biological sample, antibodies which are characteristic of and/or specific to multiple sclerosis, consisting in bringing said sample into contact with a reagent of the invention, and after having optionally detected the presence of an immune complex, in separating the latter;
a method of binding, in a biological sample, antibodies directed against the MSRV-1 virus, comprising the steps consisting is bringing said sample into contact with a reagent of the invention comprising at least one polypeptide pxMSRV-1, and after having optionally detected the presence of an immune complex, in separating the latter;
according to a preferred embodiment of any of the methods of binding of the invention, the sample is chosen from serum, cerebrospinal fluid and urine;
an immunotherapeutically active composition, in particular a vaccine preparation, comprising at least one polypeptide capable of reacting specifically with the antibodies of patients suffering from multiple sclerosis (MS) and whose peptide sequence comprises at least one sequence chosen from SEQ ID NO: 1 to SEQ ID NO: 19, and their equivalent sequences, and optionally a support for said polypeptide and/or a pharmaceutically acceptable excipient;
the use of at least one polypeptide capable of reacting specifically with the antibodies of patients suffering from multiple sclerosis (MS) and whose peptide sequence comprises at least one sequence chosen from SEQ ID NO: 1 to SEQ ID NO: 19, and their equivalent sequences, for binding, in a biological sample, antibodies which are characteristic of and/or specific to multiple sclerosis, and the use of at least one polypeptide pxMSRV-1 of the invention for binding, in a biological sample, antibodies directed against the MSRV-1 virus; and
a polynucleotide whose nucleotide sequence comprises a sequence encoding a polypeptide of the invention.